Centromeric DNA Facilitates Nonconventional Yeast Genetic

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Centromeric DNA facilitates nonconventional yeast genetic engineering Mingfeng Cao, Meirong Gao, Carmen Lorena Lopez-Garcia, Yutong Wu, Arun Somwarpet Seetharam, Andrew Josef Severin, and Zengyi Shao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00046 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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ACS Synthetic Biology

Submitted to ACS Synthetic Biology on Feb.5th, 2017

Centromeric DNA facilitates nonconventional yeast genetic engineering Mingfeng Cao1, 2, Meirong Gao1, 2, Carmen Lorena Lopez-Garcia1, 2, Yutong Wu1, Arun Somwarpet Seetharam3, Andrew Josef Severin3, and Zengyi Shao1, 2, 4, 5, * 1

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Department of Chemical and Biological Engineering NSF Engineering Research Center for Biorenewable Chemicals (CBiRC) 3 Genome Informatics Facility, Office of Biotechnology 4 Interdepartmental Microbiology Program 5 The Ames Laboratory 4140 Biorenewables Research Laboratory, Iowa State University, Ames, IA 50011, USA

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Correspondence to: Dr. Zengyi Shao, 4140 Biorenewables Research Laboratory, Iowa State University, Ames, IA 50011, USA Phone: 515-294-1132 Email: [email protected]

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Abstract Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has the highest native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S. stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extrachromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogenous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from < 1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.

Keywords Nonconventional yeasts, episomal plasmids, centromeres, CRISPR/Cas9, Scheffersomyces stipitis, CEN epigeneticity

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Saccharomyces cerevisiae is far from being the only yeast of economic importance. Many of the 1800 other known yeast species have highly unusual metabolic, biosynthetic, physiological, and fermentative capacities that make them attractive in various biotech applications. As outcomes of long-term natural evolution in particular environments, these highperformance characteristics are conferred by a network of genes via a hierarchy of regulations that are intrinsically complex, making horizontal transfer of these functions into model hosts very challenging. With the advancement of platform technologies (e.g., next-generation sequencing1 and advanced genome editing strategies2), great interest has recently emerged in engineering native hosts to directly leverage this enormous fortune inherited from nature. In general, for engineering nonconventional species, ‘stable episomal expression platforms’ and ‘precise genome-editing tools’ are the two foundational technologies. For S. cerevisiae, there are low-copy number plasmids, high-copy number plasmids, and various homologous recombination (HR)-based genome-editing tools. In contrast, for majority of the nonconventional yeast species, episomal plasmids are not available, and many nonconventional yeasts rely on non-homologous end joining (NHEJ) mechanism to repair double-strand DNA breaks in chromosomes3, 4, causing an extremely high false-positive rate when performing targeted genome modifications. The recent rapid advance of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology seems to point out a solution for this latter issue, but still suffers from a low efficiency when lacking a stable plasmid to express the CRISPR components. At the early stage of strain development, especially for nonconventional species, a stable episomal expression platform could provide unmatchable flexibility in assembling genetic information and in turn facilitate each of the downstream development steps.

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Unfortunately, the vast majority of yeast species do not have the naturally born 2µ highcopy number plasmid. To trick cells into recognizing extra-chromosomal DNA as their own genetic material, an autonomously replicating sequence (ARS) and a centromere (CEN) need to be built into the expression vector to direct the replication and segregation of the episomal DNA, respectively. An ARS is a DNA replication starting point, and hundreds of such sequences are normally found in a yeast genome5, 6. Nowadays, the technique used for ARS isolation is simple and quite mature7, 8 (Figure S1), whereas challenges remain in identifying a functional CEN in an efficient manner. We chose Scheffersomyces stipitis as the target species. S. stipitis is one of the most important microorganisms in the field of biorenewables due to its high native capacity for converting xylose9, the second most abundant sugar in lignocellulosic biomass. It has previously served as a repository for isolating genes involved in xylose transport10, 11 and utilization12-14, but recently demonstrated the potential as a well-suited microbial host for producing high-value compounds derived from shikimate pathway15 whose downstream products (mainly including flavonoids and alkaloids) have highly desired nutraceutical and pharmaceutical properties16-18. However, its direct implementation as a fermenting host is limited by the lack of the two foundational technologies as described above. Here we successfully pinpointed the centromere from the chromosome 5 in S. stipitis genome. The identified CEN sequence significantly stabilized the ARS-containing vector, enabled homogenous gene expression, and increased lactic acid production as a commercially relevant compound by 3-fold. By incorporating CRISPR components, the targeted gene-knockout efficiency was improved from less than 1% to more than 80%. This finding not only facilitates technology development for exploiting high-potential

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yeast species, but also provokes profound discussion regarding CEN-specific genome features that have been rarely recognized in the past.

Results and Discussion Instability of the S. stipitis vector lacking a CEN A CEN is the partitioning element present on each chromosome that directs the formation of a kinetochore, the multi-protein complex that interacts with the spindle microtubules to enable stable chromosome segregation during cell division19-21. In the absence of a CEN, extrachromosomal DNA containing an ARS can replicate, but is mitotically unstable and lost with a high frequency due to the strong maternal segregation bias22, 23. Such a critical instability issue was first noticed when transforming S. stipitis with the existing vector carrying an enhanced green-fluorescence protein (eGFP) expression cassette. An unusually broad eGFP fluorescence range was observed, which was in great contrast to the sharp, uniform peak when expressing eGFP in any commercial vectors developed for S. cerevisiae (Figure 1a). Fluorescence-activated cell sorting (FACS) was performed to separate cells into three groups with different levels of fluorescence. Copy number analysis of each group by real-time PCR indicated that the ARSeGFP plasmid was present at 0–140 copies per cell (Figure 1b); when high-eGFP signal cells were isolated, the plasmid dropped quickly from 140 to 6 copies on average per cell within 48 h, at which time approximately 72% of the entire population had completely lost the plasmid (corresponding to the blue peak in Figure 1a). This instability was in fact caused by the missing stabilizing element (i.e., CEN) on the episomal plasmid whose role is to guarantee stable DNA segregation during cell division. Plasmids carrying ARS but no CEN tend to stay in the mother cells during cell division, making the daughter cells randomly receive 0 to a few copies of the

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plasmids in each round. The retained plasmids in the mother cells will then be accumulated, throughout multiple rounds of cell division, to a high copy number. To the best of our knowledge, all the episomal plasmids developed for S. stipitis to date were designed based on the same backbone, varying only in the selection marker, but all missing the key CEN element24-26. It is worth noting that neither the S. cerevisiae CEN nor the 2µ origin27, 28 functioned in S. stipitis.

GC3 chromosome scanning for CEN prediction The traditional CEN isolation strategies (Figure S2) include (a) chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq)29, 30 and (b) functional selection based on toxic gene lethality at a high copy number31, 32. We failed in applying either of these two strategies because the former one sets a specific prerequisite for a centromeric protein-specific antibody and the latter one requires relatively high transformation efficiency for establishing a genomic DNA library. It is generally accepted that CEN formation is modulated in an epigenetic manner in eukaryotes, conferring a heritable phenotype that is not based solely on a genotype33, 34. To date, no cis-acting CEN-specific DNA sequences have been found conserved across yeast species, or even among chromosomes belonging to a single species. Previously it was found that the genomic GC content is correlated with meiotic recombination hot spots35, 36. In S. cerevisiae, the genes nearby centromeres and telomeres show reduced crossover, making these regions nonrandomly associated with cold spots37, 38. The evolutionary decrease in GC content is particularly more evident at wobble positions because mutations occurring here are often silent39, allowing the maintenance of a genome structure feature without affecting gene functions.

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Evidence in this regard can be found from Lynch et al.’s pure bioinformatics analysis40, where they calculated the GC percentage of the third positions of codons (GC3) on each chromosome for a group of yeasts, including Candida lusitaniae, S. stipitis, and Yarrowia lipolytica. Their results showed that the in silico prediction was effective for five of the six Y. lipolytica chromosomes; the five CENs that were experimentally pre-confirmed by other groups22, 41 coincided with the single markedly reduced GC3 trough in each chromosome. This delivered a very important message that some yeasts might contain signature ‘GC3 valleys’ featuring centromeric functions. However, due to the lack of functional verification directly following the bioinformatics prediction, this hypothesis based on in silico analysis has not been widely appreciated yet. To address the current plasmid instability issue in S. stipitis, we performed a more detailed analysis of the identified GC3 valleys. Lynch et al.’s work indicated that a uniquely sharp GC3 valley arose on each chromosome, but in fact the valleys spanned on chromosomes in a very large range from 92 kb to 194 kb in length. An unusually long intergenic region ranging from 14,594 bp to 38,042 bp was found within each valley, whereas the average length of the other intergenic regions was as short as 732 bp to 1976 bp. We hypothesized that the CENs are located within these abnormally long intergenic sequences.

Stepwise identification of the minimal CEN5 for S. stipitis The longest intergenic region from chromosome 5 GC3 valley (width: 105,828 bp) was selected based on its relatively short length (17,264 bp) (Figure 2a), and more importantly, on the observation that several genes flanking this target location are homologs of the open reading frames proximal to the CENs isolated from C. albicans and C. dubliniensis29, 40. Flow cytometry

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analysis of the yeast cells containing this cloned 17.3-kb region in the backbone of the ARSeGFP vector displayed a symmetric eGFP expression peak, with the eGFP-positive population increasing from 28% to 93% relative to the yeast cells transformed with the ARS-eGFP vector (Figure 2b). In experiments with a series of variants with shortened CENs, we found that the core CEN sequence could be reduced stepwise from 17.3 kb to 125 bp while maintaining the enhanced eGFP expression profile. The colonies with a functional CEN revealed a uniformly medium growth rate, whereas those without a CEN displayed a range of sizes, indicating a variable copy number among the cells. It was also noticed that cells harboring CENs shorter than 500 bp formed colonies much more slowly (almost 7 days on a SC-URA plate for cells with the125-bp CEN; see Figure S3). This result was likely caused by an inefficient interaction between the CEN core and the kinetochore components when the proximal regions were trimmed off, as special 3D structures need to be formed to facilitate CEN-kinetochore binding. The 500-bp fragment (named CEN5-500bp) was therefore chosen as the minimal stabilizing element for future plasmid manipulations. The paradox of CENs challenges the classic view of a genetic locus42, 43. “Point CENs” direct the formation of a single CenH3 nucleosome that connects to a single microtubule during mitosis44. To date, the vast majority of the yeast species with point CENs (e.g., S. cerevisiae, C. glabrata, Kluyveromyces lactis, and Eremothecium gossypii) are known to have ~125-bp point CENs arranged in CDEI/II/III blocks, in which the CDEI (~8 bp) and CDEIII (~25 bp) regions are separated by a non-conserved AT-rich CDEII region (78–86 bp)45. “Regional CENs” contain a large array of binding sites for centromeric proteins, forming multiple CenH3 nucleosomes attached to microtubules within a specific region of the chromosome19, 44. For example, the representative pathogenic budding yeast C. albicans contains 3–5 kb non-conserved CENs that

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assemble into kinetochores containing Cse4-rich centromeric chromatin29, whereas the CENs of the fission yeast Schizosaccharomyces pombe range from 40 to 110 kb and contain long repetitive elements45. It was previously believed that S. stipitis carries long regional CENs because of its close evolutionary relationship with Candida species, the majority of which do not have CEN-specific sequence motifs29, 40, 46. However, our findings that the 125-bp sequence that conferred the centromeric function to an ARS-containing vector strongly favors the ‘point CENs’ classification for S. stipitis. This new 125-bp sequence joined the CENs isolated most recently from Naumovozyma castellii (based on ChIP-sequencing)47 and the ones proposed for Kuraishia capsulata48, thereby challenging the conventional view that point CENs are relatively conserved in evolution by forming CDEI/II/III blocks. Since chromosome 5 of S. stipitis has a total length of 1.73 Mbp, this 125-bp element represented a 14,000-fold reduction in sequence.

Comparison with the ARS vector and additional evidences to confirm the CEN identity Copy number and mitotic stability analysis of the ARS/CEN5-500bp-eGFP plasmid showed ~3–5 copies per cell, with at least 80% of the cells being eGFP-positive; in both measurements stability was observed for at least 168 h, in sharp contrast to the rapid decline of the eGFPpositive population belonging to the cells transformed with the ARS-eGFP plasmid (Figure 3a). Note that although the copy number of the ARS-eGFP plasmid also appeared stable at 2–3 copies/cell, this number simply reflected a weighted average from a range of 0–140 copies per cell. To test the expression uniformity enabled by the CEN-containing plasmid, a putative xylose transporter (XUT1) containing a C-terminal eGFP tag was expressed, and the results were examined by confocal microscope (Figure 3b). Homogeneous expression was only observed when the fusion protein was expressed using the ARS/CEN5-500bp vector, with a distinctive

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fluorescent halo observed at the cell periphery via membranous localization of the xylose transporter. This result was in contrast to the case when the fusion protein was expressed in the ARS vector, only brightest cells could be observed. In addition, a codon-optimized lactate dehydrogenase (LDH) gene from Lactobacillus helveticus was cloned into the ARS/CEN5500bp vector, leading to lactate production at a titer of 29.5 ± 0.4 g/L, which was 3-fold higher than the level achieved with the corresponding plasmid lacking the CEN (9.4 ± 1.2 g/L, Figure 3c). This ARS/CEN5-500bp vector should be particularly useful for producing valuable chemicals in S. stipitis considering that alterations in most biosynthetic pathways interfere with cellular metabolism and therefore inhibit the hosts’ growth rate to a certain degree; an unstable plasmid would be quickly lost during cultivation. Two additional lines of evidence support the CEN identity. First, a constitutive promoter was inserted upstream of CEN5-500bp, which completely abolished the nearby CEN function (Figure 4a). This may have occurred through a mechanism involving transcriptional CEN inactivation49, 50, wherein active local transcriptional activity interrupts functional interactions between CEN and the segregation machinery. Second, while one CEN is required for stable plasmid segregation, the presence of two copies of 125-bp core CEN either in tandem or separately on a plasmid showed a detrimental effect (i.e., no transformants; Figure 4b). This is because the mitotic spindle apparatuses attached to both CENs and tore the plasmid under the opposing forces in two directions51. These observations evidently confirmed that the identified elements are indeed CENs, rather than sequences that contribute to plasmid stability through directly tempering ARS function (i.e., low-copy autonomously replicative plasmids can be maintained more stably than high-copy plasmids).

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Site-specific gene knockout facilitated by the CRISPR technology With the success in addressing the episomal plasmid instability issue, we next integrated the CRISPR technology to tackle the second major challenge faced by many nonconventional species – the difficulty in performing site-directed genome modifications, which is determined by the relative efficiency of NHEJ over HR. In S. stipitis, the dominancy of NHEJ prohibits sitespecific gene knockout. In our previous study, transforming an ura3 deletion fragment carrying various lengths of homology arms in a range of 100 bp to 1 kb failed in generating the expected ura3 knockout after screening 100 colonies that appeared on the selection plate. To create a CRISPR system for S. stipitis, the Cas9 gene had to be recoded because S. stipitis uses a nonstandard coding system and translates the CUG codon to serine instead of leucine9. We synthesized a S. stipitis-compatible codon-optimized version of Cas9 gene (Table S1), and fused the sequences encoding SV40 nuclear localization signal (NLS) at both ends (Figure 5a). The expression of Cas9 gene was enabled by the constitutive eno1 promoter and the tef1 terminator. To enable the synthesis of guide RNA, we excavated the RNA polymerase III promoter PSNR52 from S. stipitis genome by aligning the potential sequence coding the SNR52 rDNA with the ones identified from S. cerevisiae52 and Candida albicans53. Two genes, ade2 and trp1, were selected as the targets. Disruption of ade2 will confer an easily visible red phenotype in medium with a low level of adenine supplementation, whereas a trp1 knockout strain will lose the ability of tryptophan biosynthesis. For the ade2 knockout, the red colonies growing on the plate with a low concentration of 10 mg/L adenine hemisulfate supplementation indicated a knockout efficiency of higher than 80% (5 out of 6 colonies were red, Figure 5b), and sequencing results revealed that all five strains had the random indel mutations occurring at the target locus (Figure 5c). This result indicated that with the integration

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of CRISPR and stable episomal expression, gene knockout efficiency in S. stipitis was increased at least 80-fold considering the previous failure for the same purpose by transforming a deletion cassette carrying homologous arms. For the trp1 knockout, an efficiency of 100% indel mutations was achieved at the target site with the mutants exhibiting growth deficiency on the plate without tryptophan supplementation (Figure 5d and 5e). The success in integrating the stable episomal expression platform with the CRISPR-enabled genome editing highlights the main avenue for engineering nonconventional yeasts. Many of them outperform S. cerevisiae, but the exploration pace is constrained by the limited availability of genetic manipulation tools. Recently, we demonstrated that S. stipitis serves as a better-suited microbial host than S. cerevisiae for producing shikimate pathway derivatives15. Expressing the same set of enzymes accounting for shikimate production in the two hosts immediately resulted in a 7-fold difference in titer. This is primarily because S. stipitis offers higher availability of the rate-limiting precursor erythrose 4-phosphate54 owing to its much more active pentose phosphate pathway. In plants, shikimate pathway provides the precursors for synthesizing a huge panel of complex structures including flavonoids, stilbenoids, and benzylisoquinoline alkaloids9-11. These molecules are often associated with desired antioxidant, anti-inflammatory, antimicrobial, antifungal, herbicidal, or anticancer activities and thus have huge market values55-58. Development of the foundational technologies in this study therefore provides the underpinning to innovate advanced-level strategies (e.g., enhancing HR by removing the key genes responsible for NHEJ, and genome-scale transcriptional network study based on CRISPR interference and activation) to explore S. stipitis as a robust microbial factory in the near future to produce such an important and diverse class of high-value compounds. This kind of platform-technology integration holds great promise for shortening the process of tool development, leading to

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unprecedented advances in exploration of the treasures to be found in high-potential nonconventional species.

Materials and Methods GC3 chromosome scanning. The whole genome sequence of S. stipitis was downloaded from National Center for Biotechnology Information (NCBI), along with their annotations (in fasta and GFF format, respectively). The coding sequences (CDS) were then extracted from the genome using BEDTools (v2.20.1)59. CodonW (v1.4.4)60 was used to calculate the GC3 percentage for each CDS sequence and a line graph was generated with a moving average of 15 genes corresponding to each chromosome.

Plasmid construction and yeast transformation. Majority of the plasmids used in this study were constructed using the DNA assembler method developed previously61. In brief, the PCRamplified fragments with overlapping ends were co-transformed with a digested plasmid backbone into S. cerevisiae for plasmid assembly via electroporation or lithium acetate-mediated methods. The isolated yeast plasmids were then transformed into E. coli for enrichment, and their identities were verified by restriction digestion or sequencing. The correctly assembled plasmids were subsequently transformed into S. stipitis for target gene expression. Key primer sequences, codon-optimized genes, CEN-containing sequence, and plasmid maps are summarized in Supporting Information Table S1, Table S2 and Figure S4.

Flow cytometry analysis and cell sorting. The transformed S. stipitis cells were cultured in SC-URA medium for ~36–48 h and then centrifuged for 2 min at 2,000 × g to remove the

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supernatant. The cell pellets were resuspended in 10 mM phosphate-buffered saline (pH 7.4) to an optical density at 600 nm (OD600nm) between 0.1 and 0.2, and then analyzed by flow cytometry at 488 nm on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA). The fluorescence-intensity distribution of each clonal population was calculated by BD FACSCanto Clinical Software. FACS was performed on a BD FACSAria III for copy number analysis. Three groups of cells, with high-level (mean fluorescence = 77,014), medium-level (mean fluorescence = 3,430) and low-level (mean fluorescence = 329) signals were sorted; 105 cells were collected for each group and re-inoculated into SC-URA medium for time-dependent copynumber assays.

Step-wise determination of the minimal CEN5. Based on GC3 chromosome scanning results, the longest intergenic region located in the GC3-valley of chromosome 5 was hypothesized to harbor a functional CEN. To clone this 17,264-bp sequence, eight overlapping fragments, with lengths of 2.4 kb to 2.6 kb, were amplified from S. stipitis genomic DNA and assembled into the ARS-eGFP plasmid linearized by SacI and NotI using the DNA assembler method61. To ensure high assembly efficiency, 400-bp overlaps between adjacent fragments were maintained through carefully designing primer-annealing positions62, 63. The ARS/CEN5-eGFP plasmid conferred a symmetric fluorescence peak in transformed cells and was therefore deemed to contain a functional CEN. The 17,264-bp sequence was subsequently divided into three segments of ~6kb, each of which was cloned to the ARS-eGFP plasmid backbone. The segment that conferred a robust, symmetrical eGFP expression peak was continuously shortened to trim away the unnecessary sequences.

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CRISPR-based gene knockout. The codon-optimized cas9, with a FLAG tag and a pair of flanking nuclear localization sequences, was synthesized using the GeneArt service at Life Technologies (Carlsbad, CA), and expressed under the eno1 promoter and tef1 terminator. The target sequences for ade2 and trp1 were designed using the online tool CRISPRdirect (https://crispr.dbcls.jp/) and co-transcribed with the guide RNA by the SNR52 promoter. The above components were assembled into the ARS/CEN5-500 plasmid linearized by ClaI and XhoI using the DNA assembler method61, 62. 2 µg of the resulting plasmid pCasAde2 was individually electroporated to S. stipitis, the transformants were first selected on the selection medium SC-URA plates containing 55 mg/mL of adenine hemisulfate. The colonies were subsequently re-streaked on SC-URA containing 10 mg/mL of adenine hemisulfate. The plasmid pCasTrp1 was transformed using the same procedure, and the transformants were first selected on SC-URA followed by being re-streaked on SC-URA-TRP plates. The indel mutations were confirmed by sequencing the target knockout loci.

Supporting Information Tables S1-S2: lists of all the codon-optimized genes, key primer sequences, CEN-containing sequence, SNR52 promoter and sgDNA for ade2 and trp1 targets. Figures S1-S4: lists of traditional ARS and CEN isolation methods, growth of the clones carrying step-wisely optimized CEN5, and plasmid maps. The extended Materials and Methods about strains, reagents, plasmid construction and yeast transformation, yeast plasmid copy number assay, homogeneity of protein expression and lactic acid production, together with extended references supporting these experimental procedures.

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Author Contributions M. C. and Z. S. designed the entire set of experiments and wrote the manuscript. M. C. performed the experiments to identify S. stipitis CENs, verified their impact on plasmid stability, isolated the CEN core sequence and performed sequence comparison. M. G. contributed to the XUT1-eGFP expression study. C. L. L. contributed to the GC3 analysis of the chromosomes. Y. W. contributed to the transcriptional CEN inactivation experiment. A. S. S. and A. J. S wrote the code to perform GC3 chromosome scanning.

Acknowledgements This work was supported in part by Iowa Energy Center (4782024) and the National Science Foundation Grants (EEC-0813570 and EPS-1101284). We thank Dr. Thomas W. Jeffries for providing the strain S. stipitis FLP-UC7 (ura3-3, NRRL Y-21448). We also thank Dr. Shawn M. Rigby for performing flow cytometry analysis, and Dr. Margaret Carter for confocal microscope examination.

Competing Financial Interests The authors declare the following competing financial interest(s): Z. S., M. C., and M. G. declare competing financial interests in the form of a pending patent.

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Figure Legends Figure 1. Instability of the S. stipitis plasmid in the absence of a functional centromere (CEN). a. Comparison of enhanced green fluorescence protein (eGFP)-expression profiles between S. stipitis carrying the ARS-eGFP plasmid and S. cerevisiae carrying the ARS/CEN-eGFP plasmid. Blue peak: population completely losing plasmids; this population presumably relies on the secreted amino acid synthesized by the population expressing the auxotrophic amino acid marker, but cannot be re-grown in the selection medium lacking that amino acid after cell sorting. Green peak: population carrying plasmids in different copy numbers. The width of the green peak represents the variation of eGFP expression per cell, which is administrated by plasmid copy number. b. Copy number analysis over 7 days. Fluorescence-activated cell sorting (FACS) was performed to separate cells into groups with three different fluorescence levels. The analysis was performed in triplicate and the error bars represent the corresponding standard deviations.

Figure 2. Improving plasmid stability by incorporation of a functional CEN5 into the autonomously replicating sequences (ARS) backbone. a. The GC3 chromosome scanning profile of chromosome 5 in S. stipitis. The width of the GC3-valley belonging to chromosome 5 is marked as 105,828 bp, and the red dot indicates the exact CEN5 location identified by the subsequent experiments conducted in this research. b. Stepwise identification of the minimal CEN5 from S. stipitis chromosome 5. The 17.3 kb segment represents the longest intergenic region located in GC3-valley, and the blocks labelled in green are the segments carrying the functional CEN5 confirmed by eGFP expression.

Figure 3. The beneficial impact of the CEN element. a. Enhanced copy number stability. Note that although the copy number of the ARS-eGFP plasmid appeared stable at 2–3 copies/cell, this 20


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number simply reflected a weighted average from a range of 0–140 copies per cell. b. Homogeneous protein expression enabled by the ARS/CEN vector. The two images had nearly identical cell densities, and the scale was presented as 2 µm per division. c. Lactic acid production was tripled when lactate dehydrogenase (LDH) was expressed from the ARS/CEN500bp vector. All the assays were performed in triplicate and the error bars represent the corresponding standard deviations.

Figure 4. Additional lines of evidence used to confirm the CEN identity. a. Incorporation of an active promoter immediately upstream of a CEN disrupted the interactions between the segregation machinery and the CEN due to the local transcriptional activity. b. Arranging two copies of CEN on one plasmid led to plasmid breakage during plasmid segregation.

Figure 5. CRISPR-mediated gene knockout in S. stipitis. a. The construct design of expressing Cas9 in the stable ARS/CEN vector. Sc-elements contain the ura3 selection marker and the ARS/CEN sequences specific for S. cerevisiae, which were used for efficient DNA assembly; Ec-elements contain the ampicillin selection marker and the pMB1 origin of replication used for plasmid propagation in E. coli; Ss stands for S. stipitis. b. Knocking out ade2 led colonies to develop easily visible red color at a low adenine supplementation. c. Confirming the indel mutations occurring at the ade2 locus. d. Knocking out trp1 abolished the growth on the plate lacking tryptophan supply. e. Confirming the indel mutations occurring at the trp1 locus.

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Figure 1.

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Figure 2.

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Figure 3

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Figure 4.

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Figure 5.

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Graphic of Table of Content 1057x767mm (72 x 72 DPI)


Centromeric DNA Facilitates Nonconventional Yeast Genetic

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